Project summary: Current platforms of inactivated split virus (split) and live attenuated influenza virus (LAIV) vaccines based on immunity to the hemagglutinin (HA) hypervariable protein do not provide effective cross protection against antigenically distinct new strains. Developing a novel vaccine that improves the efficacy and breadth of cross protection is therefore of high priority. Previous studies aimed to generate universal influenza vaccines suggest that universal antigenic targets are insufficient to serve as a standalone vaccine due to low efficacy of protection compared to HA-matched split vaccine despite the benefits of broadening cross protection. Because of this limitation, universal influenza vaccine candidates are not expected to replace current influenza vaccine platforms. Our progress report demonstrated that a combination of commercial HA-based influenza split and M2 ectodomain (M2e) vaccines significantly improved the efficacy of cross protection compared to either M2e- or HA-based vaccine alone. Enhanced cross protection by M2e-supplemented HA vaccination was consistently observed in studies using mouse and ferret animal models. In this renewal application, we propose groundbreaking approaches to increase the intrinsic capacity of current influenza vaccine platforms (LAIV, Split) to confer cross protection by incorporating conserved M2e epitopes into HA in a chimeric form. In our preliminary studies, we rescued recombinant influenza viruses expressing a chimeric HA molecule with conserved M2e epitopes (4xM2e-HA, M2e-HA) using reverse genetics. More importantly, these recombinant influenza virus vaccines with chimeric HA containing M2e epitopes could confer significantly enhanced protection against a broader range of viruses (H1, H3, and H5 subtypes) by inducing cross protective M2e antibodies without compromising HA immunity. In aim 1, we will test the hypothesis that recombinant seasonal (H1N1 and H3N2) LAIV and inactivated split vaccines with chimeric HA containing M2e epitopes will enhance the efficacy of cross protection by inducing immunity to both M2e and HA. Replication competent recombinant seasonal H1N1 and H3N2 influenza viruses will be constructed by engineering chimeric M2e-HA molecules. Aim 2 will investigate the contribution of cellular and humoral immune mechanisms to cross protection by recombinant influenza virus vaccines using wild type and mutant mouse models. Aim 3 will validate the efficacy of cross protection by recombinant influenza virus vaccines in ferrets, the most relevant small-animal model for the assessment of influenza vaccines.